The C-Terminal Disulfide Bonds of Helicobacter pylori GroES Are Critical for IL-8 Secretion via the TLR4-Dependent Pathway in Gastric Epithelial Cells

Abstract

Helicobacter pylori GroES (HpGroES), a potent immunogen, is a secreted virulence factor that stimulates production of proinflammatory cytokines and may contribute to gastric carcinogenesis. HpGroES is larger than other bacterial orthologs because of an additional C-terminal region, known as domain B. We found that the HpGroES-induced IL-8 release by human gastric epithelial cells was dependent on activation of the MAPK and NF-κB pathways. HpGroES lacking domain B was unable to induce IL-8 release. Additionally, a TLR4 inhibitor significantly inhibited IL-8 secretion and reduced HpGroES-induced activation of MAPKs. Furthermore, HpGroES-induced IL-8 release by primary gastric epithelial cells from TLR4−/− mice was significantly lower than from wild-type mice. We also found that HpGroES bound to TLR4 in cell lysates and colocalized with TLR4 on the cell membrane only when domain B was present. We then constructed two deletion mutants lacking C-terminal regions and mutants with point mutations of two of the four cysteine residues, C111 and C112, in domain B and found that the deletion mutants and a double mutant lacking the C94–C111 and C95–C112 disulfide bonds were unable to interact with TLR4 or induce IL-8 release. We conclude that HpGroES, in which a unique conformational structure, domain B, is generated by these two disulfide bonds, induces IL-8 secretion via a TLR4-dependent mechanism.

Importance of the C-terminal histidine residues of Helicobacter pylori GroES for Toll-like receptor 4 binding and interleukin-8 cytokine production

“…Previous studies have demonstrated that Hp GroES binds TLR4 on the cell surface25. To further establish the binding abilities of WT and mutant proteins to TLR4 on the cell surface, KATO-III cells were incubated with 0.05 μM WT or histidine mutants, and the localization of Hp GroES and TLR4 determined via confocal microscopy.…”

“…The cytokine plays a role in gastric mucosal injury caused by H. pylori and is correlated with the development of chronic gastritis16. Domain B of Hp GroES contributes to Hp GroES-induced IL-8 secretion through a TLR4-dependent mechanism25. Domain B displays a unique conformational loop structure generated by two disulfide bonds referred to as the four-cysteine ring (4CR) motif.…”

“…Hp GroES was also identified as a dominant gastric cancer-related antigen with significantly higher seropositivity in gastric cancer patients, compared with gastritis and duodenal ulcer patients24. The recently characterized, mechanism of Hp GroES-induced IL-8 production by gastric carcinoma cells involves activation of the MAPK and NF-κB pathways via a TLR4-dependent mechanism25.…”

“…This metal binding ability is associated with nickel homeostasis and protection of cells from higher concentrations of nickel ion272830. In addition, Hp GroES binds TLR4 on the cell membrane only in the presence of domain B. Hp GroES lacking the disulfide bonds of domain B is unable to interact with TLR4 and induce IL-8 production25. Mutational analyses of the Cys residues in domain B revealed that these residues are crucial for interaction with the TLR4 receptor.…”

“…We previously demonstrated that domain B of Hp GroES contributes to GroES-induced IL-8 secretion through a TLR4-dependent signaling pathway25. It was shown that a synthetic peptide corresponding to the cysteine /histidine rich sequence of domain B Hp GroES has high affinity to nickel ions31.…”